Project Summary The long term goal of this project is to develop, validate and commercialize a PCR-based molecular test for routine Alzheimer?s disease (AD) testing that will provide more comprehensive risk estimates for disease and offer a superior option for clinical trial assays that can help accelerate drug development. This test anticipates the utility of a powerful risk marker in the TOMM40 gene (rs10524523 or ?523) that is difficult to genotype accurately due to a variable length poly-T repeat. The novel, single-tube workflow will combine the determination of repeat polymorphisms at this locus with the detection of well-established Apolipoprotein E (APOE) risk alleles to improve AD risk estimates. The proposal is innovative in that it addresses the analytical challenges associated with the analysis of the high homology poly-T polymorphism in TOMM40 to achieve reliable single-base resolution and define reliable thresholds for risk assessment, and combines this method with genotyping of the three common APOE risk alleles ?2, ?3 and ?4. Taken together, this test will produce risk estimates for the vast majority of the population rather than just those with ?4. This work addresses unmet needs for more informative tools to help predict the onset of AD and inform the management of at-risk individuals and/or those with mild cognitive impairment that may progress to AD. Once available, the test is expected to improve clinical outcomes by enabling benefits from early treatment. The test will also be useful as a clinical research tool to glean the full impact of TOMM40 and APOE on AD and other neurodegenerative disorders, and to help support emerging targeted therapeutics and clinical trials. Aim 1: Combine the detection of both APOE and TOMM40 into a rapid and simple single test assay using Asuragen?s novel AmplideX technology in conjunction with toehold hairpin primers. Aim 2: Verify the combined APOE and TOMM40 assay using well-characterized cell-line DNA, defined synthetic templates, and 50 matched and blinded blood and buccal samples. Future work will further the development these assays by incorporating controls, interpretative software, and integration consistent with a diagnostic system for risk assessment with a path to an IVD or companion diagnostic for an AD therapeutics. The outcome of the assay development will be new tools that can also incorporate patient-specific information to improve risk assessments and inform links between genotype and neurodegenerative phenotype.